Synthesis of steroids



United States Patent 1 Claim. (Cl. '195-51) ABSTRACT OF THE DISCLOSUREPreparation of 4,8,l4-trimethyl 18 nor-8a,9,8,14[3-androst-4-en-3,11,17-trione by subjecting 4oz,8,l4trimethyl-l8-nor-5a,8a,9fi,145-androstane-3,l1,17-trione to Pseudomonastestosteroni.

This application is a division of our application Ser. No. 442,202,filed Mar. 23, 1965, and now US. Patent 3,320,290.

This invention relates to and has for its object the provision of a newphysiologically active compound, and more particularly,4a,8,14-trimethyl-18-nor-8a,9,B,146- androst-4-en-3,11,17-trione.

The novel compound of this invention is a pharmacologically activesubstance which possesses anti-androgenic activity (i.e., they inhibitthe actions of androgens) and which may be used in the treatment of suchconditions as hyperandrogenic acne. It also possesses antiestrogenic andanti-gonadotrophic activity.

The compound may be formulated for such administration, theconcentration and/or dosage being based on the activity of theparticular compound and the requirements of the patient.

The final product of this invention is prepared by the process of thisinvention which entails beginning with 40:,8,l4 trirnethyl 18 nora,8cz,9}3,14fi-&11dl'08t3116 3,11,17-trione as a starting material. Thepreparation of this compound is disclosed in copending US. patent ap'plication Ser. No. 442,203, now US. Patent 3,316,281, filed on an evendate with this application. It has been found that the compound of thisinvention can be pre: pared from the starting material by subjecting thelatter to the action of a microorganism of the genus pseudomonas or tothe action of the enzymes thereof under oxidizing and preferably aerobicconditions.

To prepare the compounds of this invention, the starting material may befirst subjected to the action of enzymes of a microorganism of the genuspseudomonas under oxidizing conditions. This oxidation can best beeffected either by including the starting material in an aerobic cultureof the microorganism, or by bringing together in an aqueous medium, thecompounds, air, and enzymes of non-proliferating cells of themicroorganism.

In general, the conditions of culturing the pseudomonas microorganismfor the purposes of this invention are (except for the inclusion of thestarting material to be converted), the same as those of culturingvarious other microorganisms for the production of antibiotics, vitaminB-l2, and other like substances. The microorganism is grown aerobicallyin contact with (in or on), suitable fermentation medium. A suitablemedium essentially comprises a source of carbon and energy. The lattermay be a carbohydrate, for example, molasses, glucose, maltose, starchor dextrin, a fatty acid, a fat and/ or the compound itself. Preferably,however, the medium includes an assimilable source of carbon and energyin addition to the 3,373,084 Patented Mar. 12, 1968 steroid. Among thefats utilizable for the purpose of this invention are lard oil, soybeanoil, linseed oil, cottonseed oil, peanut oil, coconut oil, corn oil,castor oil, sesame oil, crude palm oil, fancy mutton tallow, sperm oil,olive oil, tristearin, tripalrnitin, triolein and trilaurin. Among thefatty acids utilizable for the purpose of this invention are stearicacid, palmitic acid, oleic acid, linoleic acid and m-yristic acid.

The source of nitrogenous factors utilizable for the purposes of thisinvention may be organic (e. g., soybean meal, cornsteep liquor, yeastextract, meat extract and/or distillers solubles) or synthetic (i.e.,composes of simple, synthesizable organic or inorganic compounds, suchas ammonium salts, alkali nitrates, amino acids or urea).

An adequate sterile air supply should be maintained during fermentation,for example, by the conventional methods of exposing a large surface ofthe medium to air or by utilizing submerged aerated culture. Thecompound may be added to the culture during the incubation period, orincluded in the medium prior to sterilization or inoculation. Thepreferred (but not limiting) range of the concentration of the compoundin the culture is about 0.01% to about 0.1%. The culture period (orrather the time of subjecting the compound to the action of the enzyme)may vary considerably, the range of about twenty-four to ninety-sixhours being feasible, but not limiting.

The microbial process described hereiuabove yields 411,8, l4trimethyl-l8-nor-8ot,913,l4;3-androst-4-en-3,l1,17- trione.

The invention may be illustrated by the following example, alltemperatures are in degrees centigrade unless otherwise stated.

EXAMPLE 1 Surface growth from each of 4 two-week-old agar slants ofPseudomonas testosteroni ATCC (American Type Culture Collection) No.11996, the slants containing as a nutrient medium (A):

Distilled water to 1 liter.

is suspended in 5 ml. of 0.01% aqueous sodium lauryl solution. One ml.portions of this suspension are used to inoculate sixteen 250 ml.Erlenmeyer flasks, each containing 50 ml. of the following sterilizedmedium (B):

Grams Beef extract 1.5 Yeast extract 3 Peptone 6 Dextrose 1 Distilledwater to 1 liter 1 After 18 hours incubation at 25 C. with continuousrotary agitation (280 cycles/minute; two inch radius), 5% (v.:v.)transfers are made to one hundred 250 ml. Erlenmeyer flasks, eachcontaining 50 ml. of freshly sterilized medium B. After 24 hours offurther incubation, using the same conditions described above, eachflask is supplemented with 200 micrograms/ml. of 4m,8,l4-trimethyl 18nor-5a,8u,9fi,14 3-androstane-3,l1,17-trione. The steroid is added bysupplementing each flask with 0.25 ml. of a sterile solution (40mg./ml.) of a steroid in N,N-dimethyl-formamide. A total of 1.0 gm. iffermented. After 6 days of further incubation, using the same conditionsas described above, the contents of the flasks are 3 pooled and thebroth is then adjusted to pH 4.0 using 12 N H 50 The acidified broth isthen filtered through a Seitz clarifying pad. The flasks mycelium andpad are washed with successive 50 ml. portions of warm water. Thecombined filtrate and washings have a volume of 5700 ml. They areextracted three times with 1900 ml. portions of chloroform which arecombined, Washed twice with 2 liter portions of water and evaporated, invacuo. The residue is plate chromatographed on Woelm neutral alumina(Activity V) using chloroform as the developing solvent. After elutingethyl acetate and crystallizing from 4 acetone-hexane, the product40;,8,14-trimethyl-l8-nor- 8a,9fi,14;3-androst-4-en-3,l 1,17-trione isobtained.

What is claimed is:

1. A process for the preparation of4,8,14-trimethyll8-nor-8a,9,8,14B-androst-4-en-3,11,17-trione whichcomprises subjecting 4:1,8,14-trimethyl-18-nor-5u,8a,9;8,145-

androstane-3,11,17-trione to the action of a microorgan-l ism of thegenus Pseudomonas testosteronit No references cited.

ALVIN E. TANENHOLTZ, Primary Examiner.

